CN112877343B - Barley stripe disease pathogenicity gene Pgr03723 and application thereof - Google Patents

Abstract

The application provides a barley stripe disease pathogenicity gene Pgr03723 and application thereof, wherein the gene is derived from wheat nucleophile Pyrenophora graminea. The barley stripe disease belongs to a systemic infectious fungal disease, is one of main diseases of barley, and pathogenic bacteria of the barley stripe disease mainly have sexual stage and asexual stage, are distributed in various barley planting areas of the world, and are seriously damaged in river basin areas such as Shanghai, sichuan, zhejiang and Jiangsu. The Pgr03723 interference mutant strain is obtained through an RNA interference technology, and the gene is further provided to participate in regulating and controlling the growth and differentiation of the strain, so that theoretical and practical significance is provided for elucidating the pathogenic mechanism of barley stripe bacteria, enriching the gene function of plant pathogenic bacteria, novel disease-resistant control strategies and disease-resistant breeding of barley.

Description

Barley stripe disease pathogenicity gene Pgr03723 and application thereof

Technical Field

The application belongs to the field of molecular biological engineering, and in particular relates to bioinformatics analysis of barley stripe germ pathogenicity gene Pgr03723 and mycelium growth rate regulation and control thereof; in addition, an RNAi technology is adopted to construct an interference vector of the gene, and an interference strain is obtained through genetic transformation, so that the application of pathogenicity of barley streak can be reduced.

Background

The barley stripe disease (Pyrenophora graminea) belongs to systemic infectious fungal diseases, is one of main diseases of barley, and pathogenic bacteria mainly have sexual phases and asexual phases, wherein the sexual phases are called rhizoctonia cerealis [ Pyrenophora graminea (rank.) Ito et Kurib ], belong to ascomycotina and sclerotium, and the asexual phases are called Podoptera littoralis [ Drechslera graminea (Rabenh.ex Sch 1.) ], belong to Desmokatzia; the asexual stage is common in nature, and the sexual stage is rare. When the pathogenic bacteria infects the leaf blades or leaf sheaths of the barley, the conidium is generally columnar, light yellow to yellow brown, most straight, hemispherical at two ends, generally pointed at the widest end at the basal cell, 1-7 diaphragms with the size of 50-125 mu m multiplied by 14-25.5 mu m, generally forms secondary conidium peduncles at the apical cell or the basal cell, has obvious navel points and is deeply sunk in the basal cell. Conidiophores extend from the stomata, are single or few clusters, are columnar straight or bent, are not branched, and are light brown to brown.

At present, the core technology for developing pathogenic research and breeding of disease-resistant varieties is mainly used for identifying the disease resistance of plant germplasm resources. Because of the pressure of natural selection, the co-evolution of disease resistance genes of host plants and pathogenic genes of pathogens, different barley streak strains often show toxicity differences to the same disease resistance gene, so that barley streak has stepped differences in hypha morphology, physiological characteristics and pathogenicity. The primary research on the barley on the stripe disease aims to know the interaction relation of barley and stripe disease pathogens under different environmental conditions and how the barley generates the regularity of disease resistance, so that the occurrence of the barley stripe disease is effectively prevented, and the quality and the yield of the barley are improved.

The research takes the barley stripe disease strong pathogenicity strain QWC as a material, takes a genome sequencing result as a basis, screens out and clones the barley stripe disease effector Pgr03723, researches the functions of the barley stripe disease strong pathogenicity strain Pgr03723 by an RNAi method, verifies the related functions of the barley stripe disease strong pathogenicity strain Pgr 5734 from the aspects of the form, microstructure and pathogenicity of hyphae, and defines the functional mechanism of pathogenicity of effector protein Pgr03723 in barley stripe bacteria, thereby providing theoretical and practical significance for elucidating the pathogenic mechanism of the barley stripe bacteria, enriching the gene function of plant pathogenic bacteria, novel barley disease resistance control strategy and disease resistance breeding.

Problems of the prior art: no report on the application of barley stripe disease (Rhizoctonia cerealis) Pgr03723 gene and RNA interference of the gene is found in the prior art.

Disclosure of Invention

In view of the shortcomings of the prior art, the application provides a pathogenic gene Pgr03723 of barley stripe disease (barley nucleophile), which is prepared by using an RNA interference technology to obtain Pgr03723 interference mutant according to the requirements of the disease target gene, and verifying the related functions from the aspects of the form, microstructure and pathogenicity of hyphae, and the functional mechanism of pathogenicity of effector protein Pgr03723 in barley stripe bacteria is clear, so that the aim is achieved, the application adopts the following technical scheme:

1. pathogenic gene Pgr03723 of barley stripe disease (barley nucleophile), the full-length sequence of which is shown in SEQ ID NO: 1.

2. A method for acquiring pathogenic genes Pgr03723 of barley streaks (barley nucleophiles), comprising:

(1) Culture of barley stripe strain

PDA medium was prepared: weighing 200g of fresh potatoes, peeling and removing bud eyes, cutting into small cubes of 2-4cm <2 >, adding 1.2L of distilled water, boiling for 20-30min, filtering to remove residues by using gauze, adding 20g of glucose and 17g of agar into filtrate, stirring and mixing uniformly by using a glass rod, adding distilled water to a volume of 1L, sterilizing by high-pressure steam at 121 ℃ for 20min, counting down plates in an ultra-clean workbench when the culture medium reaches 50 ℃, airing the culture medium completely, taking a bacterial cake at the edge of a barley stripe germ strain QWC stored at 4 ℃ by using a puncher with the diameter of 5-10mm, and placing the bacterial cake on a PDA culture medium plate for 7d.

(2) Cloning of Pgr03723 Gene

Cloning Pgr03723 gene by taking cDNA of a genome of a barley stripe germ wild strain QWC strain as a template, carrying out PCR amplification by using a primer Pgr03723-F1/Pgr03723-R1 (shown in figure 1), purifying a product, connecting 1 mu L of a carrier pMD19-T Vector (Simple), 5 mu L of a target fragment 4 mu L, solution I, slowly mixing, lightly centrifuging for 5sec, connecting overnight at 16 ℃), transforming E.coli Trans1-T1 competent cells (preparing LBA solid culture medium plates containing 100 mu g/mL ampicillin (Amp), uniformly coating 80 mu L X-gal and 20 mu L IPTG on each plate, airing for later use, taking out E.coli Trans1-T1 competent cells stored at-80 ℃, dissolving on ice, adding 5 mu L of a connecting liquid every 25 mu L competent cells, lightly and uniformly agitating the LB, rapidly heating at 42 ℃, carrying out sealing at 90 ℃ for 3 mu L of ice bath, carrying out shaking for 1 mu L of a sealing culture medium, carrying out filtration for 2 mu L of a sealing culture medium, and carrying out 2 mu L of sealing for 2h in a sealed culture medium, and carrying out PCR (shake for 2h, and carrying out 2 h).

(3) Pgr03723 gene sequences and bioinformatics analysis

RNA of a wild strain QWC is extracted and reversely transcribed into cDNA, a pMD19-T Vector (Simple) Vector is connected through PCR amplification (shown in figure 2A), competent cells of escherichia coli are transformed, and then cDNA sequences of Pgr03723 genes are obtained through sequencing, and the cDNA sequences are successfully cloned into Pgr03723 genes of the Nuclear bacteria (P.graminea). Bioinformatics analysis is carried out on Pgr03723 genes, and on-line software is used for prediction analysis, so that the result is obtained: analysis of the full length 1239bp of the Pgr03723 gene according to the ORFfinder software, translation of the Pgr03723 code 413 amino acids by the ExPASy software, and prediction analysis of physicochemical properties by the ProtParam software: pgr03723 encodes a protein having an isoelectric point of about 6.09, which is an acidic protein; the molecular weight Mw of the protein is 43.51KD, the content of alanine (Ala) in the amino acid composition is 14.3% at most, and the content of glycine (Gly) in the amino acid composition is 7.7%; the average hydrophilicity (GRAVY) is-0.019; the total number of positively charged residues (Arg+Lys) was 33 and the total number of negatively charged residues (Asp+Glu) was 36; the fat index (aliphatics index) is 74.99; the molecular formula is C1940H2996N504O591S21, and the total number of atoms is 6052; the instability index (instability index, II) was 36.87, giving the protein a stable (II <40, i.e. stable protein). The protein secondary structure was predicted and analyzed using SOPMA tool, with Random coil (Cc) at a maximum of 47.94% and Beta-turn (Tt) at a minimum of 5.81%, alpha helix (Hh) at 29.54% and Extended strand (Ee) at 16.71%. (FIG. 2B); the protein was analyzed for hydrophilicity by ProtScale software, peaks above 0 indicated that the protein was hydrophobic, and peaks below indicated that the protein was hydrophilic, as inferred by combining the fat index (aliphatics index) and the average hydrophilicity value (grady), pgr03723 encoded protein hydrophilic protein (fig. 2C); the NetPhos3.1 Server software predicts and analyzes the phosphorylation site to: the Pgr03723 gene encodes proteins with Serine (Ser), tyrosine (Tyr) and Threonine (Threonine, thr) phosphorylation sites (FIG. 2D); TMpred software predicts the protein transmembrane region to give the presence of the transmembrane region for Pgr03723 encoded protein, which is predicted to belong to the transmembrane protein (fig. 2E).

The application of Pgr03723 gene in the aspects of barley stripe germ growth differentiation, pathogenicity and the like comprises the following steps:

(1) Construction of the interference vector pSilent-1Pgr03723

The cDNA of strain QWC was used as a template, and the primers Pgr03723-F1, pgr03723-R1, and Pgr03723-F2, and Pgr03723-R2 (FIG. 1) were used to amplify the fragments Pgr03723-1 (492 bp) and Pgr03723-2 (492 bp), respectively. The fragment Pgr03723-1 was double-digested with KpnI and Bgl II and the vector pSilent-1, and the fragment pSilent-1 was ligated into the vector pSilent-1 using T4 ligase. Then, the fragments Pgr03723-2 and pSilent-1Pgr03723-1 were digested with XhoI and HindIII, and the fragment Pgr03723-2 was ligated to the vector pSilent-1Pgr03723-1 using T4 ligase to give the interfering vector pSilent-1Pgr03723 of the Pgr03723 gene (FIG. 3).

(2) Screening of genetic transformants

Placing wild strain QWC strain in enzymolysis solution (10 mg/ml muramidase+20 mg/ml cellulase R-10, formulated with 1.2mol/L NaCl stabilizer) at 30deg.C, filtering the sterile solution, re-suspending, shaking the suspension at 30deg.C at 80rpm/min for 3-4 hr, filtering the suspension into 10m centrifuge tube, centrifuging at 1996rpm/min at room temperature for 5min,washed twice with 1.2mol/L NaCl and finally resuspended in NTC (1.2M NaCl+10mM Tris-HCl (pH=7.5) +10mM CaCl) ₂ ) Preserving in solution. Transformation by PEG4000 mediation: taking 100 mu LNTC to re-suspend and precipitate, adding about 25 mu g of carrier plasmid pSilent-Pgr03723 connected with target fragments, gently mixing, standing at normal temperature for 20min, sequentially adding 200 mu L, 200 mu L and 800 mu LPTC, and slowly mixing uniformly every time PTC is added. Then standing at room temperature for 15min, adding 3mL of rPD culture medium, mixing gently, and finally standing and culturing for 24h in a 25 ℃ incubator. After stationary culture, the mixture is divided into two parts, and then is evenly mixed with 20mL of rPDA culture medium respectively, poured into a culture dish, and after complete solidification, the culture dish is sealed and placed in a culture box at 25 ℃ for culture. Carefully observing the surface of an rPDA plate, if visible colonies are formed, adding 20mL of PDA culture medium containing 50 mug/mL hygromycin B into an ultra-clean workbench, sealing the culture medium plate by a sealing film, continuously culturing in a dark environment at 25 ℃, observing the plate at regular time until the capped rPDA grows single colonies, respectively picking the colonies by a white gun head, inoculating the colonies into a new PDA culture medium plate containing 50 mug/mL hygromycin B, continuously culturing and screening the colonies, continuously subculturing for three generations, inoculating a transformant strain capable of stably growing into the PDA culture medium plate for culturing, rapidly extracting genome DNA by using a reagent for rapidly lysing fungal microorganisms to release nucleic acid, performing PCR identification and screening by using a hygromycin B specific primer HYG-F/HYG-R, and performing fluorescent quantitative PCR analysis to obtain interference ratios of the following substances respectively: 66.04%, 64.61%, 23.31%, 57.80% and 59.64% of the required primers are shown in FIG. 1.

(3) Identification of pathogenicity of mutant strains

Selecting the strain Pgr03723-1 and QWC (CK) strain with the largest interference rate, inoculating the strain into a PDA culture medium, culturing for 7d in the dark 25, infecting the barley seeds with QWC and the interference strain for 20d at 6 ℃ by adopting a sandwich method, and calculating and observing the morbidity and the morbidity after 20d of potting sowing. The strain infects isolated barley leaves, the leaves are obviously diseased at the infested part of a wild strain QWC bacterial cake, lesions appear, the leaves are yellow brown lesions, and the attacks of the infected leaves of the interfering strain are weaker. The morbidity of the wild strain QWC and the interference strain Pgr03723-1 are respectively: 100% and 31.66% (FIG. 4), the incidence of the interfering strain Pgr03723-1 was significantly reduced compared to the control. In conclusion, the gene Pgr03723 of the barley stripe bacteria strain is related to pathogenicity and participates in pathogenicity of the wheat rhizoctonia strain.

Drawings

FIG. 1 shows the sequences of the primers used in the present application.

FIG. 2 shows a diagram of Pgr03723 gene bioinformatics analysis.

Wherein A is the amplification result and the sequence comparison diagram of the Pgr03723 gene, B is the protein secondary structure prediction diagram of the Pgr03723 gene, C is the hydrophilicity-hydrophobicity diagram of the Pgr03723 gene, D is the phosphorylation site diagram of the Pgr03723 gene, and E is the transmembrane region prediction diagram of the Pgr03723 gene.

FIG. 3 is a schematic diagram of Pgr03723 gene interference vector.

FIG. 4 is a graph showing the relative expression level, incidence and morphology of the interfering strain and the wild strain.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the following detailed description of the embodiments of the present application will be given with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the application shown in the drawings and described in accordance with the drawings are merely exemplary and the application is not limited to these embodiments. It should be noted here that, in order to avoid obscuring the technical solution of the present application due to unnecessary details, only the structures and/or processing steps closely related to the solution according to the present application are shown in the drawings, while other details having little relation are omitted.

Example 1

The embodiment provides a barley stripe disease pathogenicity gene Pgr03723, which is derived from barley stripe bacteria (Pyrenophora graminea), and the CDS sequence of the gene is shown in a sequence table 1.

Example 2

The embodiment provides a method for cloning barley stripe disease pathogenic gene Pgr03723 and obtaining RNA interference transformant thereof, comprising the following steps:

1. cloning of Pgr03723 Gene and bioinformatic analysis thereof

PDA medium was prepared: weighing 200g of fresh potatoes, peeling and cutting the potatoes into small cubes, adding 1.2L of distilled water, boiling for 20-30min, filtering residues by using gauze, adding 20g of glucose and 17g of agar into the filtrate, stirring and mixing uniformly, adding distilled water to a volume of 1L, sterilizing by high-pressure steam at 121 ℃ for 20min, counting down plates in an ultra-clean workbench when the culture medium reaches 50 ℃, taking out bacterial cakes from the edges of the bacterial strains QWC of barley stripe bacteria preserved at 4 ℃ by using a puncher with the diameter of 5-10mm after the culture medium is completely aired, and placing the bacterial cakes on the plates of PDA culture medium for 7d.

Cloning Pgr03723 gene by taking cDNA of a genome of a barley stripe germ wild strain QWC strain as a template, carrying out PCR amplification by using a primer Pgr03723-F1/Pgr03723-R1 (shown in figure 1), purifying a product, connecting 1 mu L of a Vector pMD19-T Vector (Simple), 5 mu L of a target fragment 4 mu L, solution I, slowly mixing, lightly centrifuging for 5sec, connecting overnight at 16 ℃), transforming E.coli Trans1-T1 competent cells (preparing LBA solid medium plates containing 100 mu g/mL ampicillin (Amp), uniformly coating 80 mu L X-gal and 20 mu L IPTG on each plate, airing for later use, taking out DH5 mu L competent cells stored at-80 ℃, dissolving on ice, adding 5 mu L of a connecting liquid every 25 mu L competent cell, lightly and elastically mixing LB, carrying out ice bath 30min,42 ℃ heat shock 90s, rapidly carrying out stable operation, carrying out shaking for 1 mu L of a liquid culture medium at 3 ℃ and 200 mu L of a white medium, carrying out shaking for 1 mu L of a 2h in a sealed culture box, and carrying out 2h shaking for 1-37 h in a sealed culture box, and carrying out filtration.

RNA of a wild strain QWC is extracted and reversely transcribed into cDNA, a pMD19-T Vector (Simple) Vector is connected through PCR amplification (shown in figure 2A), competent cells of escherichia coli are transformed, and then cDNA sequences of Pgr03723 genes are obtained through sequencing, and the cDNA sequences are successfully cloned into Pgr03723 genes of the Nuclear bacteria (P.graminea). Bioinformatics analysis is carried out on Pgr03723 genes, and on-line software is used for prediction analysis, so that the result is obtained: analysis of the full length 1239bp of the Pgr03723 gene according to the ORFfinder software, translation of the Pgr03723 code 413 amino acids by the ExPASy software, and prediction analysis of physicochemical properties by the ProtParam software: pgr03723 encodes a protein having an isoelectric point of about 6.09, which is an acidic protein; the molecular weight Mw of the protein is 43.51KD, the content of alanine (Ala) in the amino acid composition is 14.3% at most, and the content of glycine (Gly) in the amino acid composition is 7.7%; the average hydrophilicity (GRAVY) is-0.019; the total number of positively charged residues (Arg+Lys) was 33 and the total number of negatively charged residues (Asp+Glu) was 36; the fat index (aliphatics index) is 74.99; the molecular formula is C1940H2996N504O591S21, and the total number of atoms is 6052; the instability index (instability index, II) was 36.87, giving the protein a stable (II <40, i.e. stable protein). The protein secondary structure was predicted and analyzed using SOPMA tool, with Random coil (Cc) at a maximum of 47.94% and Beta-turn (Tt) at a minimum of 5.81%, alpha helix (Hh) at 29.54% and Extended strand (Ee) at 16.71%. (FIG. 2B); the protein was hydrophilic by analysis with ProtScale software, peaks above 0 indicated the protein as hydrophobic, peaks below indicated the protein as hydrophilic, and by combining the fat index (aliphatics index) and the average value of hydrophilicity (grady) it was deduced that the protein encoded by Pgr03723 was a hydrophilic protein (fig. 2C); the NetPhos3.1 Server software predicts and analyzes the phosphorylation site to: the Pgr03723 gene encodes proteins with Serine (Ser), tyrosine (Tyr) and Threonine (Threonine, thr) phosphorylation sites (FIG. 2D); TMpred software predicts the protein transmembrane region to give the presence of the transmembrane region for Pgr03723 encoded protein, which is predicted to belong to the transmembrane protein (fig. 2E).

2. Construction of the interference vector pSilent-1Pgr03723

The cDNA of strain QWC was used as a template, and the primers Pgr03723-F1, pgr03723-R1, and Pgr03723-F2, and Pgr03723-R2 (FIG. 1) were used to amplify the fragments Pgr03723-1 (492 bp) and Pgr03723-2 (492 bp), respectively. The fragment Pgr03723-1 was double-digested with KpnI and Bgl II and the vector pSilent-1, and the fragment pSilent-1 was ligated into the vector pSilent-1 using T4 ligase. Then, the fragments Pgr03723-2 and pSilent-1Pgr03723-1 were digested with XhoI and HindIII, and the fragment Pgr03723-2 was ligated to the vector pSilent-1Pgr03723-1 using T4 ligase to give the interfering vector pSilent-1Pgr03723 of the Pgr03723 gene (FIG. 3).

3. Screening of genetic transformants

The wild strain QWC strain was incubated in an enzymatic hydrolysate (10 mg/ml lywallzyme+20 mg/ml cellulase R-10, formulated with 1.2mol/L NaCl stabilizer) at 30℃for 3h, the sterile solution was filtered and resuspended, the suspension was shaken at 80rpm/min at 30℃for 3-4h, the suspension was filtered into a 10m centrifuge tube, centrifuged at 1996rpm/min at room temperature for 5min, washed twice with 1.2mol/L NaCl, and finally resuspended in NTC (1.2M NaCl+10mM Tris-HCl (pH=7.5) +10mM CaCl2) solution for storage. Transformation by PEG4000 mediation: taking 100 mu LNTC to re-suspend and precipitate, adding about 25 mu g of carrier plasmid pSilent-Pgr03723 connected with target fragments, gently mixing, standing at normal temperature for 20min, sequentially adding 200 mu L, 200 mu L and 800 mu LPTC, and slowly mixing uniformly every time PTC is added. Then standing at room temperature for 15min, adding 3mL of rPD culture medium, mixing gently, and finally standing and culturing for 24h in a 25 ℃ incubator. After stationary culture, the mixture is divided into two parts, and then is evenly mixed with 20mL of rPDA culture medium respectively, poured into a culture dish, and after complete solidification, the culture dish is sealed and placed in a culture box at 25 ℃ for culture. Carefully observing the surface of an rPDA plate, if visible colonies are formed, adding 20mL of PDA culture medium containing 50 mug/mL hygromycin B into an ultra-clean workbench, sealing the culture medium plate by a sealing film, continuously culturing in a dark environment at 25 ℃, observing the plate at regular time until the capped rPDA grows single colonies, respectively picking the colonies by a white gun head, inoculating the colonies into a new PDA culture medium plate containing 50 mug/mL hygromycin B, continuously culturing and screening the colonies, continuously subculturing for three generations, inoculating a transformant strain capable of stably growing into the PDA culture medium plate for culturing, rapidly extracting genome DNA by using a reagent for rapidly lysing fungal microorganisms to release nucleic acid, performing PCR identification and screening by using a hygromycin B specific primer HYG-F/HYG-R, and performing fluorescent quantitative PCR analysis to obtain interference ratios of the following substances respectively: 66.04%, 64.61%, 23.31%, 57.80% and 59.64% of the required primers are shown in FIG. 1.

Example 3

The embodiment provides an application of barley stripe disease pathogenic gene Pgr03723 in regulating and controlling pathogenicity of wheat-like nucleophile, comprising:

selecting the strain Pgr03723-1 and QWC (CK) strain with the largest interference rate, inoculating the strain into a PDA culture medium, culturing for 7d in the dark 25, infecting the barley seeds with QWC and the interference strain for 20d at 6 ℃ by adopting a sandwich method, and calculating and observing the morbidity and the morbidity after 20d of potting sowing. The strain infects isolated barley leaves, the leaves are obviously diseased at the infested part of a wild strain QWC bacterial cake, lesions appear, the leaves are yellow brown lesions, and the attacks of the infected leaves of the interfering strain are weaker. The morbidity of the wild strain QWC and the interference strain Pgr03723-1 are respectively: 100% and 31.66% (FIG. 4), the incidence of the interfering strain Pgr03723-1 was significantly reduced compared to the control. In conclusion, the gene Pgr03723 of the barley stripe bacteria strain is related to pathogenicity and participates in pathogenicity of the wheat rhizoctonia strain.

The foregoing is merely illustrative of the embodiments of this application and it will be appreciated by those skilled in the art that variations and modifications may be made without departing from the principles of the application, and it is intended to cover all modifications and variations as fall within the scope of the application.

<110> Gansu agricultural university

<120> barley streak disease pathogenic gene Pgr03723 and application thereof

<160>1

<210>1

<211>1242bp

<212> DNA

<213> barley stripe bacteria-Rhizoctonia cerealis (Pyrenophora graminea)

<400>

1 ATGCATTTCA CTAGCTCCCC TCTCTACTTC GCCGTCGTTG GCTTATCATC CTCATCTGCT

61 GTCCTTGCAG GTGTCCTGCC CTACGCACAA TGCGGTGGCA AGACCTTCAA GGGTGATAGC

121 ACCTGCGCTG AGGGCTGGTC TTGTGTCAAG ATCAACGACT GGTACAGCCA GTGTGTTCCA

181 GTTCCTGCTT GCACTGCTGG CCCGACTGAC CCGACCTTCA ACGCCACTCT CCCAGTCGCC

241 ACGCCTGCTG CGGTTGTCTC GCCCACACCT GCTGCCTCTC CCGCCAACAA CTCTGCCCCC

301 GCACCCAACG TTGCCGGCAA CGGCGCCAAC GGCGCCAAGT GCAACCTCGA TGCCGTCATG

361 AAGGCCAAGG GTAAGAAGTA CCTTGGTGTC GCCACCGACC AAGGCCTCCT CACCCGCGAC

421 AAGAACGCCG ACATCGTCAA GGCCAACTTT GGTTGCGTCA CCCCTGAGAA CAGCATGAAG

481 TGGGATGCCA CCGAAGGCAC CCAGGGCCAA TTCACCCTTT CCGGTGCCAA CTACCTCGTC

541 GACTTTGCTA CCAAGAATGA CAAGCTGGTC CGTGGCCACA CCACCGTCTG GCACTCGCAA

601 CTCCCTACCT GGGTCTCCTC CATCACCGAC AAGACCAAGC TCACCGAGGT CATGGTCGCC

661 CATATCAAGA AGCTCATGAC CACCTACGCC GGCAAGGTCT ACGCCTGGGA CGTAGTCAAC

721 GAGATCTTCG CCGAAGAAGG CGGTTTCCGC TCCTCAGTCT TCTACAATGT TCTCGGCGAA

781 GACTTTGTCG CTACCGCCTT CGCAGCTGCC AAAGCCGCCG ACCCAGAGGC TAAACTCTAC

841 ATCAACGACT ACAACCTTGA CAGCCCCAGC TACGCAAAGA CAAAGGCCAT GGCATCCCAC

901 GTCAAGAAGT GGATCGCCGC TGGTGTTCCC ATCGACGGCA TCGGCTCCCA GTCCCATCTT

961 TCTGGCGTCT GGCCCATGTC TGATGTTCCT GCTGCTATGG AGCTTCTCTG CGGTTCCGCC

1021 CCCGAATGCG CAATGACTGA GCTCGACATC AAGGGTGGAG CGGCAAAGGA CTACAAGACT

1081 GCCTTTGACG CTTGCCTGAA CCAGAAGAAC TGTGTCGGCG TTACCATTTG GGGTGTTAGC

1141 GACAAGACCT CATGGATTGG CGCTGCTGCT ACCCCGTTGT TGTTCGATAA TAGCTTCCAG

1201 GCTAAGCCTG CTTACAACGA GCTTTGCTCG GCGCTTGCTT AA

Claims (1)

1. The application of Pgr03723 gene in reducing pathogenicity of wheat nucleophile is characterized in that the full-length sequence of Pgr03723 is shown in SEQ ID NO:1 is shown in the specification; the application of reducing the pathogenicity of the sclerotinia gracilis is realized by adopting the interference Pgr03723 gene expression.